The objective of this project is to isolate and study the cellular components involved in the interconversion of phylloquinone and phylloquinone-2,3-epoxide as well as the vitamin K-dependent carboxylation of glutamic acid residues in the precursor protein to form gamma-carboxyglutamic acid found in active prothrombin. Studies of the enzymes and other factors involved in these processes will be designed to determine if epoxidation of vitamin K is coupled to the carboxylation of glutamic acid or is involved in some other vitamin K-dependent step in the biosynthesis of prothrombin. The possible role of vitamin K in the carboxylation of protein other than those involved in blood coagulation will also be studied. The specific research goals are: (a) To determine the function of the cytosolic protein and NADH in epoxidation, prothrombin synthesis and carboxylation of glutamic acid in the precursor protein. (b) To determine the nature of the energy requirement in prothrombin synthesis and the mechanism of CO2 activation in the carboxylation reaction. (c) To determine the nature of the differences in reactivity when the quinone or hydroquinone of vitamin K is used as substrate for epoxidation and prothrombin synthesis. (d) To continue experiments designed to isolate the active components of phylloquinone epoxidase and phylloquinone epoxide reductase from the microsomal fraction of rat liver. (e) To continue to study the effect of anticoagulants on the interconversion of phylloquinone and its 2,3-epoxide to determine on a molecular level the mechanism by which these compounds inhibit prothrombin synthesis. (f) To study the possibility that vitamin K and its epoxidation may be involved in other carboxylation reactions not associated with clotting protein synthesis.